luxel mitoxpress xtra stress single

MitoXpress® Stress Test Mitochondrial Stress Test

Companion Kit to be used in combination with Luxcel’s MitoXpress® Xtra - Oxygen Consumption Assay

ILLUMINATING DISCOVERY®

CONTENTS GENERAL INFORMATION ............................................................. 3 MATERIALS SUPPLIED ...................................................................... 3 ADDITIONAL ITEMS REQUIRED ....................................................... 3 OPTIONAL ITEMS (NOT SUPPLIED) ................................................. 3 SUPPORT .......................................................................................... 3

DESCRIPTION .................................................................................. 4 TYPICAL WORKFLOW ..................................................................... 5

PERFORMING THE MITOXPRESS® XTRA STRESS TEST........... 6 CELL CULTURE AND PLATING .......................................................... 6 PRE-ASSAY PREPARATION ............................................................... 7

TYPICAL ASSAY............................................................................... 8 ANALYSIS ........................................................................................ 10 MITOCHONDRIAL STRESS TEST....................................................... 10 SUBSTRATE DEPLETION LIMITS MAXIMAL RESPIRATION................ 12

APPENDIX A - EXPLANATORY NOTES..................................... 13 REFERENCES ........................................................................... 14 RELATED PRODUCTS ............................................................. 14

GENERAL INFORMATION MATERIALS SUPPLIED Assay kit will arrive at room temperature. Store individual components as indicated below. The kit is designed for use with MitoXpress® Xtra Oxygen Consumption Assay (MX-200).

Cat. #

Comp.#

Kit Component

Desc.

Contents

Storage

No of Wells1

MXC-600

MXC-601

Oligomycin

1 vial

10 µg

-20°C

55

MXC-602

FCCP

1 vial

4 µg

-20°C

55

MXC-603

Antimycin A

1 vial

3 µg

-20°C

55

MXC-604

Glucose Oxidase

1 vial

112.5 µg

-20°C

6

Number of wells based on described procedure, stock dilutions and concentrations. Using triplicates, 18 complete MitoXpress® Xtra Stress Tests can be performed with the metabolic modulators included in this kit.

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NOTE: On receipt, each MitoXpress® Xtra Stress Test component should be stored as indicated above. A labelled zip-lock bag is provided for convenient storage at -20°C.

ADDITIONAL ITEMS REQUIRED • MitoXpress® Xtra Oxygen Consumption Assay Kit (MX-200) • Standard clear 96- well TC+ plates OR 96- well black wall clear bottom TC+ plates • Fluorescence plate reader with suitable filters and plate temperature control OPTIONAL ITEMS (NOT SUPPLIED) • Plate block heater for plate preparation SUPPORT • Visit our website www.luxcel.com (all user manuals are available for download from the website).

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DESCRIPTION Oxygen consumption measurements have become a key functional readout of cell metabolism and mitochondrial function, providing important insights into the cell function and role of perturbed metabolism in disease progression. These measurements are easily performed in standard microplates using Luxcel’s MitoXpress® Xtra Oxygen Consumption Assay.

While measurements of Basal Oxygen Consumption alone are very informative, additional information can be gleaned from assessing parameters such as Basal Respiration, Maximal Respiration, Spare Respiratory Capacity, ATP-Coupled, Non ATP-Coupled and Non-Respiratory Oxygen Consumption. These parameters are now conveniently measurable on conventional fluorescence plate readers with the development of Luxcel’s MitoXpress® Xtra Stress Test.

Designed for use in combination with MitoXpress® Xtra Oxygen Consumption Assay, the MitoXpress® Stress Test contains all metabolic modulators required to characterise the main parameters of mitochondrial function in live cells. Compounds are provided pre-weighed for convenient handling and reproducibility. A detailed protocol is provided to facilitate easy and reproducible data generation and interpretation.

Reducing equivalents generated from the TCA drive the electron transport chain (ETC) to generate a proton gradient across the inner mitochondrial membrane, with O2 being the terminal electron acceptor. F1/FO ATPase uses the proton gradient to power ATP production.

Oligomycin blocks this process, revealing the level of ATP-Coupled Oxygen Consumption.

FCCP dissipates the proton gradient and consequently increases Oxygen Consumption to reveal Maximum and Spare Respiratory Capacity.

ETC activity is blocked by Antimycin A revealing Non-Respiratory Oxygen Consumption.

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Figure 1: Overview of mitochondrial respiration and effects of metabolic modulators included in this kit

TYPICAL WORKFLOW

DAY 1

Plate cells and return to culture overnight

Prepare MitoXpress速 Stress Test controls Prepare MitoXpress速 Xtra reagent (MX-200)

Aspirate spent media DAY 2

Add fresh media and MitoXpress速 Xtra reagent Add control compounds at pre-optimised concentrations Overlay plate with HS mineral oil (MX-200)

Measure on fluorescence plate reader using recommended settings Analyse kinetic data output as recommended

Figure 2: Typical MitoXpress速 Stress Test Workflow

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PERFORMING THE MITOXPRESS® XTRA STRESS TEST NOTE: Prior to using the MitoXpress® Xtra Stress Test, ensure that measurement settings and cell number have been optimised as per the MitoXpress® Xtra Oxygen Consumption Assay User Manual.

CELL CULTURE AND PLATING Count cells and adjust to the desired plating density in culture medium (typically ~4-8×104 cells per well for adherent cells, depending on cell type). Return the plate to culture overnight (typically >14 h). For suspension cells, seed ~4x105 cells in 100 µL medium on the day of the assay. See Appendix A – Explanatory Notes for more information.

NOTE: Do not add cells to row H (see Fig. 3 for recommended plate map); these wells will be used as Blank wells and for the Signal Controls.

PRE-ASSAY PREPARATION Prepare Kit Components: • Oligomycin (pink cap): To prepare 300 μM Oligomycin stock (200X) dissolve vial contents in 35 μL of DMSO. Aliquot into two equal volumes and store at -20°C, each aliquot is sufficient for a minimum of 25 wells (1.5 μM final concentration/well).

• FCCP (white cap): To prepare 500 μM stock dissolve vial contents in 35 μL (200X) of DMSO. Aliquot into two equal volumes and store at -20°C, each aliquot sufficient for a minimum of 25 wells (2.5 μM final concentration/well). See Appendix A for further information.

• Antimycin A (clear cap): To prepare 200 μM Antimycin stock, dissolve vial contents in 35 μL (200X) of DMSO. Aliquot into two equal volumes and store at -20°C, each aliquot is sufficient for a minimum of 25 wells (1 μM final concentration/well).

• Glucose Oxidase (green cap): Reconstitute in 70 μL distilled water to obtain 1.5 mg/mL stock solution. Aliquot into two equal volumes and store at -20°C. Each aliquot is sufficient for at least 2 wells (10 μL per well).

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Prepare on Day of Measurement: • MitoXpress® Xtra reagent (MX-200): Prepare as recommended in MitoXpress® Xtra User Manual (MX-200) by reconstituting vial content in 1 mL distilled water.

Reconstituted product can be aliquoted and stored at -20°C. Use within one month (avoid freeze thaw).

• Prepare Working Stocks from Oligomycin, FCCP and Antimycin A aliquoted as described under ‘Prepare Kit Components’ by diluting them 1/20 in fresh assay medium.

• Test compounds/drugs (optional, not provided): Dilute so that volume of added compound is kept low (1-5 μL) to minimize any potential vehicle effects. We recommend the use of triplicate wells for each treatment.

Component

Function

Stock Concentration

Recommended Final Concentration/Well

Cap Colour

Oligomycin

Inhibits F1/FO-ATPase

300 μM (200X)

1.5 μM

pink

FCCP

Uncouples ETC and F1/FO-ATPase activity

500 μM (200X)

2.5 μM

white

Antimycin A

Inhibits mitochondrial oxygen consumption

200 μM (200X)

1 μM

clear

Glucose Oxidase

Cell-free signal control

1.5 mg/mL (10X)

150 μg/mL

green

Table 1: Summary of components, recommended stock solutions and dilutions

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TYPICAL ASSAY NOTE: Plate reader, plate block heater and buffers should be pre-warmed to 37°C prior to use. Set up plate reader as described in the MitoXpress® Xtra Oxygen Consumption Assay User Manual (MX-200). For first time users, performing the signal optimisation step is advised. All user manuals are available for download from our website www.luxcel.com.

STEP 1: Remove spent culture medium from all assay wells and replace with 80 μL of fresh culture media (see Figure 3 for recommended plate map), being careful not to dislodge cells.

Add 90 μL of pre-warmed fresh culture medium to the wells designated as Signal and Glucose Oxidase Control wells (typically H 7-10).

NOTE: We recommend always leaving two wells (H 11 and H 12) free from the addition of MitoXpress® Xtra reagent, for use as Blank Controls. Add 100 μL of fresh culture media to these Blank Control wells.

STEP 2: Add 10 μL of reconstituted MitoXpress® Xtra reagent to each well, except those wells for use as Blank Controls. NOTE: If plating a full 96-well plate, we recommend combining Step 1 and Step 2 by adding the 1 mL of reconstituted MitoXpress® Xtra reagent to 10 mL pre-warmed fresh culture media and using a multi-channel pipette add, 90 μL of MitoXpress® Xtra in media stock to each well see Figure 3). Add 100 μL of fresh culture media only (no MitoXpress® Xtra) to the Blank Control wells.

STEP 3 (optional): The MitoXpress® Xtra Stress Test can be used to determine the impact of different treatments (drug treatment, signaling pathway modulation, genetic manipulation, the addition of alternative nutrients etc.) on mitochondrial respiration. Add test compound, vehicle or other desired treatment to test wells and return the plate to culture for the desired period or test immediately. For a complete characterization, all controls (Vehicle Control, Oligomycin, FCCP, and Antimycin A) should be applied for each condition.

NOTE: Keeping volume of added compound low (1-5 μL) and use of a vehicle control is recommended to minimize any potential vehicle effects.

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STEP 4: Add 10 μL of Oligomycin working stock (prepared as described above) to wells to measure ATP-Coupled Respiration (typically wells A-G 4-6, see Fig. 3), 10 μL FCCP working stock to wells to measure Maximal Respiration (typically wells A-G 7-9, see Fig. 3) and 10 μL Antimycin A working stock to wells to measure Non-Respiratory Oxygen Consumption (typically wells A-G 10-12, see Figure. 3). Add 10 μL of fresh culture medium to wells designated ‘Probe only’ (H 9-10).

Figure 3: Recommended plate map for the MitoXpress® Xtra Stress Test with optional different treatments. Using this plate map, up to 7 different conditions can be tested simultaneously in triplicate across all treatment conditions (see Appendix A for note on edge effects).

STEP 5: Add 10 μL reconstituted Glucose Oxidase to the Signal Control wells (typically H 7-8).

STEP 6: Seal each well with 100 μL of pre-warmed HS Mineral Oil, taking care to avoid bubbles.

Read the plate immediately on a fluorescence plate reader as described in the MitoXpress® Xtra Oxygen Consumption Assay User Manual.

NOTE: See MitoXpress® Xtra Oxygen Consumption Assay User Manual for additional details and tips on instrument settings, oil addition and data analysis.

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NOTE: Data processing is described in the MitoXpress® Xtra Oxygen Consumption Assay User Manual. All user manuals are available for download from our website www.luxcel.com.

MITOCHONDRIAL STRESS TEST The slopes (m) derived from the linear part of the MitoXpress® Xtra Signal Profile of cells treated with the kit components are indicative of oxygen consumption rates (OCR); an established measure of mitochondrial function.

Non-Respiratory Oxygen Consumption Rate (OCR): m Antimycin A Slope (m) of Signal Profile after Antimycin A treatment. This value can be subtracted from all other rates in many experimental designs. For further explanation see Appendix A – Explanatory notes.

Fluorescence Signal (au) or Lifetime (µs)

ANALYSIS

53

FCCP treated 48

Untreated

43

38

Oligomycin treated m

33

28

Antimycin A treated

0

10

20

30

t/min

40

50

60

Figure 4: Signal Profiles of HepG2 oxygen consumption obtained using the MitoXpress® Stress Test in combination with the MitoXpress® Xtra Oxygen Consumption Assay. Slopes (m) calculated from the linear portion of these Signal Profiles are reflective of the OCR under the imposed by the kit components and are used to determine the key characteristics of aerobic respiration (see Fig. 5) as described in the text.

Basal Respiration Rate: m Untreated – m Antimycin A Slope (m) of Signal Profile of untreated cells. Impacted by factors such as current cellular ATP demand, metabolic phenotype and substrate availability Maximal Respiration Rate: m FCCP – m Antimycin A Slope of Signal Profile after FCCP treatment. When using optimised FCCP concentrations, this value characterises the theoretical maximal mitochondrial capacity under given conditions.

FCCP dissipates the mitochondrial membrane potential, uncoupling oxygen consumption from ATP production and allowing the respiratory chain to work at maximal capacity.

Spare Respiratory Capacity: m FCCP – m Untreated Difference between Signal Profile of FCCP treated cells (Maximal Respiration Rate) and P10

untreated cells (Basal Respiration Rate). Spare Respiratory Capacity is a measure of the cells ability to respond to sudden increases in energy demand or other metabolic stressors. Reduced Spare Respiratory Capacity can be important indicator of mitochondrial dysfunction.

Non ATP-Coupled Oxygen Consumption (‘Proton Leak’): m Oligomycin – m Antimycin A Slope of Signal Profile of Oligomycin treated cells indicates dissipation of the mitochondrial proton-gradient by means other than F1/Fo-ATPase activity (see Appendix A for details). Elevated levels of Non ATP-Coupled Oxygen Consumption can be a sign of impaired mitochondrial membrane integrity. In most cases, however, dissipation is a controlled process, caused for example by ion or substrate cycling or by specialized uncoupling proteins [1] and can make up to 40% of the basal OCR in some tissues [2].

ATP-Coupled Oxygen Consumption: m Untreated – m Oligomycin Difference between Signal Profile slopes of untreated cells (Basal Respiration Rate) and Oligomycin treated cells (Non ATP-Coupled Oxygen Consumption). Reflects the portion of Basal Respiration that is linked to ATP production. a) 40

25

25

20 15 10 5 0

20 Basal

Basal Respiration Respiration

15 10 5

b)

40

40

30

OCR (µs/h)

30

b)

35

35

30

OCR (µs/h)

OCR (µs/h)

35

Maximal Maximal RespirationRespiration

Non ATP-Coupled Non ATP-Coupled O2 Consumption O2 Consumption

0 Untreated Oilgomycin FCCP Oilgomycin Untreated

25 20 15 10

Non-Respiratory Non-Respiratory O2 Consumption O2 Consumption Antimycin A FCCP Antimycin A

5 0

35

Maximal Respiration

30

OCR (µs/h)

a) 40

Maximal Respiration Spare Respiratory Capacity

25 20

Spare Respiratory Capacity

15

Basal Respiration 10

5 0

Basal Respiration

ATP-Coupled O2 Consumption ATP-Coupled O2 Consumption Non ATP-Coupled Non ATP-Coupled O2 Consumption O2 Consumption

Figure 5: Full characterisation of mitochondrial function using the MitoXpress® Stress Test in combination with the MitoXpress® Oxygen Consumption Assay. a) The OCR (m) from the linear portion of the Signal Profile from each Stress Test condition (see Figure 4) reflect Basal Respiration, Non ATP-Coupled Oxygen Consumption, Maximal Respiration and Non Respiratory Oxygen Consumption. ATP-Coupled Oxygen Consumption and Spare Respiratory Capacity are calculated from these values as described in detail in the text. b) The contribution of each of these discrete metabolic processes to the Maximal Respiration can be conveniently visualised producing a detailed picture of aerobic metabolism.

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SUBSTRATE DEPLETION LIMITS MAXIMAL RESPIRATION Substrate availability is an important contributor to Spare Respiratory Capacity. Figure 6 shows the Respiration of HepG2 cells measured in high glucose, low glucose and in low glucose plus oleate. Limited Spare Respiratory Capacity (Maximal Respiration minus Basal Respiration) is observed in the low glucose condition due to a restriction in the supply of reducing equivalents to the ETC. The provision of the dietary long chain fatty acid oleate significantly increases Spare Respiratory Capacity, as beta oxidation now provides the necessary reducing equivalents. Similar increases are achieved by increasing glucose concentration.

30

OCR (Îźs/h)

25 20

Spare Respiratory Capacity

15 10

ATP-Coupled O2 Consumption

5 0

Non ATP-Coupled O2 Consumption 0.1mN Glucose

0.1mN Glucose + Oleate

25mM Glucose

Figure 6: Basal Respiration and Maximal Respiration in HepG2 cells measured in different nutrient conditions. In low glucose medium, Spare Respiratory Capacity is low, but significantly increases in the presence of high glucose concentrations or 150 ÂľM Oleate, due to the provision of reducing equivalents to fuel ETC activity.

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APPENDIX A - EXPLANATORY NOTES MitoXpress® Xtra - Oxygen Consumption Assay The MitoXpress® Stress Test is designed as a Companion Kit for use in combination with Luxcel’s MitoXpress® Xtra Oxygen Consumption Assay (MX-200). The Oxygen Consumption Assay User Manual describes instrument set-up, assay optimisation, data analysis and troubleshooting. The described instrument set-up and signal optimisation steps should be performed prior to running a MitoXpress® Stress Test. All user manuals are available for download from our website www.luxcel.com.

Cell Plating Typically, cells are seeded at a density to achieve full confluence on the day of measurement.

Plating density, cell type, and basal metabolic rate will determine the oxygen consumption rate measured. If edge effects are observed, we recommend allowing the plate to stand for 30 min at RT after plating before returning to culture [3] of cells or to omit the outer wells of the plate.

Optimising concentrations for assay controls and test compounds NOTE: FCCP exhibits a bell-shaped dose-response which can vary between cell types.

The concentration which delivers maximum respiratory activity should be determined for each cell type. This can be achieved by running an FCCP serial dilution (typically between 15 – 0.5 μM).

Oligomycin and Antimycin A are typically used at 1.5 µM or 1 µM, respectively. However, the optimal concentration can be cell-type dependent. Optimum concentrations can be determined by measuring OCR at varying Oligomycin and Antimycin A concentrations.

Test compounds/Drugs Drugs or test compounds are typically added immediately prior to measurement. Longer treatment times can be used as required.

Non-Respiratory Oxygen Consumption In most cell types, treatment with Antimycin A will result in diminished OCRs. However, in some cases oxygen consumption is observed even though ETC has been fully blocked. The remaining oxygen consumption may be attributed to detoxifying or desaturase enzyme activity or non-respiratory NADPH oxygenase activity. If unsure, if ETC blockage with Antimycin A was achieved, optimise Antimycin A concentrations by running a serial dilution or co-treatment with Rotenone. In most cases, Non-Respiratory Oxygen Consumption can be regarded as constant and subtracted from all other values for further analysis.[4] P13

REFERENCES 1.

Jastroch, M., et al., Mitochondrial proton and electron leaks. Essays In Biochemistry, 2010. 47: p. 53-67.

2.

Brown, G.C., P.L. Lakin-Thomas, and M.D. Brand, Control of respiration and oxidative phosphorylation in isolated rat liver cells. European Journal of Biochemistry, 1990.

192(2): p. 355-362.

3.

Lundholt, B.K., K.M. Scudder, and L. Pagliaro, A simple technique for reducing edge effect in cell-based assays. Journal of Biomolecular Screening, 2003. 8(5): p. 566-70.

4.

Brand, Martin D. and David G. Nicholls, Assessing mitochondrial dysfunction in cells.

Biochemical Journal, 2011. 435(Pt 2): p. 297-312.

RELATED PRODUCTS • MitoXpress® Xtra Oxygen Consumption Assay (Cat No. MX-200) • Mitochondrial Stress Test Combi Kit (Cat No. MX-220) includes both MitoXpress® Xtra Oxygen Consumption Assay and MitoXpress® Stress Test

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Luxcel Biosciences Ltd Hoffmann Park Inchera Little Island Cork T45 YX04 T:+353 (0) 21 242 8880 E: sales@luxcel.com www.luxcel.com

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